Blood
The morphologically recognizable and functionally capable cells circulating in blood include erythrocytes, neutrophilic, eosinophilic, and basophilic granulocytes, B-lymphocytes, T-lymphocytes, nonB-lymphocytes, nonT-lymphocytes, and platelets. These mature cells derive from and are replaced, on demand, by morphologically recognizable dividing precursor cells for the respective lineages such as erythroblasts for the erythrocyte series, myeloblasts, promyelocytes and myelocytes for the granulocyte series, and megakaryocytes for the platelets.
Hematopoietic Stem and Progenitor Cells
The precursor cells derive from more primitive cells that can simplistically be divided into two major subgroups: stem cells and progenitor cells. The definitions of stem and progenitor cells are operational and depend on functional rather than on morphological criteria. Stem cells have extensive self-renewal or self-maintenance capacity. Some of the stem cells differentiate upon need, but some stem cells or their daughter cells produce other stem cells to maintain the precious pool of these cells. Thus, in addition to maintaining their own kind, pluripotential stem cells are capable of differentiation into several sublines of progenitor cells with more limited self-renewal capacity or no self-renewal capacity. These progenitor cells ultimately give rise to the morphologically recognizable precursor cells. The progenitor cells are capable of proliferating and differentiating along one, or more than one, of the myeloid differentiation pathways.
Stem and progenitor cells make up a very small percentage of the nucleated cells in the bone marrow, spleen, and blood. About ten times fewer of these cells are present in the spleen relative to the bone marrow, with even less present in the adult blood. As an example, approximately one in one thousand nucleated bone marrow cells is a progenitor cell; stem cells occur at a lower frequency.
Reconstitution of the hematopoietic system has been accomplished by bone marrow transplantation. Lorenz and coworkers showed that mice could be protected against lethal irradiation by intravenous infusion of bone marrow (Lorenz, 20 E., et al., 1951, J. Natl. Cancer Inst. 12:197-201). Later research demonstrated that the protection resulted from colonization of recipient bone marrow by the infused cells (Lindsley, D. L., et al., 1955, Proc. Soc. Exp. Biol. Med. 90:512-515; Nowell, P. C., et al., 1956, Cancer Res. 16:258-261; Mitchison, N. A., 1956, Br. J. Exp. Pathol. 37:239-247; Thomas, E. D., et al., 1957, N. Engl. J. Med. 257:491-496). Thus, stem and progenitor cells in donated bone marrow can multiply and replace the blood cells responsible for protective immunity, tissue repair, clotting, and other functions of the blood. In a successful bone marrow transplantation, the blood, bone marrow, spleen, thymus and other organs of immunity are repopulated with cells derived from the donor.
Bone marrow has been used with increasing success to treat various fatal or crippling diseases, including certain types of anemias such as aplastic anemia (Thomas, E. D., et al., Feb. 5, 1972, The Lancet, pp. 284-289), Fanconi's anemia (Gluckman, E., et al., 1980, Brit J. Haematol. 45:557-564; Gluckman, E., et al., 1983, Brit J. Haematol. 54:431-440; Gluckman, E., et al., 1984, Seminars in Hematology:21 (1):20-26), immune deficiencies (Good, R. A., et al., 1985, Cellular Immunol. 82:36-54), cancers such as lymphomas or leukemias (Cahn, J. Y., et al., 1986, Brit. J. Haematol. 63:457-470; Blume, K. J. and Forman S. J., 1982, J. Cell. Physiol. Supp. 1:99-102; Cheever, M. A., et al., 1982, N. Engl. J. Med. 307(8):479-481), carcinomas (Blijham, G., et al., 1981, Eur. J. Cancer 17(4):433-441), various solid tumors (Ekert, H., et al., 1982, Cancer 49:603-609; Spitzer, G., et al., 1980, Cancer 45:3075-3085), and genetic disorders of hematopoiesis.
Bone marrow transplantation has also recently been applied to the treatment of inherited storage diseases (Hobbs, J. R., 1981, Lancet 2:735-739), thalassemia major (Thomas, E. D., et al., 1982, Lancet 2:227-229), sickle cell disease (Johnson, F. J., et al., 1984, N. Engl. J. Med. 311:780-783), and osteopetrosis (Coccia, P. F., et al., 1980, N. Engl. J. Med. 302:701-708) (for general discussions, see Storb, R. and Thomas, E. D., 1983, Immunol. Rev. 71:77-102; O'Reilly, R., et al., 1984, Sem. Hematol. 21(3):188-221; 1969, Bone-Marrow Conservation, Culture and Transplantation, Proceedings of a Panel, Moscow, Jul. 22-26, 1968, International Atomic Energy Agency, Vienna; McGlave, P. B., et al., 1985, in Recent Advances in Haematology, Hoffbrand, A. V., ed., Churchill Livingstone, London, pp. 171-197).
Present use of bone marrow transplantation is severely restricted, since it is extremely rare to have perfectly matched (genetically identical) donors, except in cases where an identical twin is available or where bone marrow cells of a patient in remission are stored in a viable frozen state. Even in such an autologous system, the danger due to undetectable contamination with malignant cells, and the necessity of having a patient healthy enough to undergo marrow procurement, present serious limitations. Except in such autologous cases, there is an inevitable genetic mismatch of some degree, which entails serious and sometimes lethal complications. These complications are two-fold. First, the patient is usually immunologically incapacitated by drugs beforehand, in order to avoid immune rejection of the foreign bone marrow cells (host versus graft reaction). Second, when and if the donated bone marrow cells become established, they can attack the patient (graft versus host disease), who is recognized as foreign. Even with closely matched family donors, these complications of partial mismatching are the cause of substantial mortality and morbidity directly due to bone marrow transplantation from a genetically different individual.
Peripheral blood has also been investigated as a source of stem cells for hematopoietic reconstitution (Nothdurtt, W., et al., 1977, Scand. J. Haematol. 19:470-471; Sarpel, S. C., et al., 1979, Exp. Hematol. 7:113-120; Ragharachar, A., et al., 1983, J. Cell. Biochem. Suppl. 7A:78; Juttner, C. A., et al., 1985, Brit. J. Haematol. 61:739-745; Abrams, R. A., et al., 1983, J. Cell. Biochem. Suppl. 7A:53; Prummer, O., et al., 1985, Exp. Hematol. 13:891-898). In some studies, promising results have been obtained for patients with various leukemias (Reiffers, J., et al., 1986, Exp. Hematol. 14:312-315 (using cryopreserved cells); Goldman, J. M., et al., 1980, Br. J. Haematol. 45:223-231; Tilly, H., et al., Jul. 19, 1986, The Lancet, pp. 154-155; see also To, L. B. and Juttner, C. A., 1987, Brit. J. Haematol. 66:285-288, and references cited therein); and with lymphoma (Korbling, M., et al., 1986, Blood 67:529-532). It has been implied that the ability of autologous peripheral adult blood to reconstitute the hematopoietic system, seen in some cancer patients, is associated with the far greater numbers of circulating progenitor cells in the peripheral blood produced after cytoreduction due to intensive chemotherapy and/or irradiation (the rebound phenomenon) (To, L. B. and Juttner, C. A., 1987, Annot., Brit. J. Haematol. 66:285-288; see also 1987, Brit. J. Haematol. 67:252-0253, and references cited therein). Other studies using peripheral blood have failed to effect reconstitution (Hershko, C., et al., 1979, The Lancet 1:945-947; Ochs, H. D., et al., 1981, Pediatr. Res. 15(4 Part 2):601).
Studies have also investigated the use of fetal liver cell transplantation (Cain, G. R., et al., 1986, Transplantation 41(1):32-25; Ochs, H. D., et al., 1981, Pediatr. Res. 15(4 part 2):601; Paige, C. J., et al., 1981, J. Exp. Med. 153:154-165; Touraine, J. L., 1980, Excerpta Med. 514:277; Touraine, J. L., 1983, Birth Defects 19:139; see also Good, R. A., et al., 1983, Cellular Immunol. 82:44-45 and references cited therein) or neonatal spleen cell transplantation (Yunis, E. J., et al., 1974, Proc. Natl. Acad. Sci. U.S.A. 72:4100) as stem cell sources for hematopoietic reconstitution. Cells of neonatal thymus have also been transplanted in immune reconstitution experiments (Vickery, A. C., et al., 1983, J. Parasitol. 69(3):478-485; Hirokawa, K., et al., 1982, Clin. Immunol. Immunopathol. 22:297-304).
Cryopreservation Solutions and Techniques
Freezing has long been used to preserve living cells, such as blood cells, after they have been removed or separated from a donating organism. The cryopreservation and recovery of living cells, however, has proven to be quite troublesome. Cells are subjected to relatively harsh conditions during both the freezing and thawing cycles involved in the cryopreservation of cells, resulting in a low survivability rate.
Freezing is destructive to most living cells. As the external medium freezes cells attempt to maintain equilibrium and lose water, thus increasing intracellular solute concentration, until intracellular freezing occurs at about minus 10.degree.-15.degree. C. It is generally believed that both intracellular freezing and solution effects are responsible for cell injury. For example, it has been proposed that freezing destruction from extracellular ice is essentially a plasma membrane injury resulting from osmotic dehydration of the cell.
Substantial time and effort has been expended in an effort to maximize the viability of thawed cells. Such efforts have generally focused upon the development of cryoprotective agents and establishing optimal cooling rates.
Cryoprotection by solute addition is thought to occur by two potential mechanisms: (i) intracellular; by reducing the amount of ice formed within the cell; and/or (ii) extracellular; by decreasing water flow out of the cell in response to a decreased vapor pressure caused by the formation of ice in the solute surrounding the cells.
Different optimal cooling rates have been described for different cells. Various groups have looked at the effect of cooling velocity or cryopreservatives upon the survival or transplantation efficiency of frozen bone marrow cells and red blood cells (Lovelock, J. E. and Bishop, M. W. H., 1959, Nature 183:1394-1395; Ashwood-Smith, M. J., 1961, Nature 190:1204-1205; Rower A. W. and Rinfret, A. P., 1962, Blood 20:636; Rowe, A. W., and Fellig, J., 1962, Fed. Proc. 21:157; Rowe, A. W., 1966, Cryobiology 3(1):12-18; Lewis, J. P., et al., 1967, Transfusion 7(1):17-32; Rapatz, G., et al., 1968, Cryobiology 5 (1):18-25; MaZur, p., 1970, Science 168:939-949; Mazur, P., 1977, Cryobiology 14:251-272; Rowe, A. W. and Lenny, L. L., 1983, Cryobiology 20:717; Stiff, P. J., et al., 1983, Cryobiology 20:17-24; Gorin, N. C., 1986, Clinics in Haematology 15(1):19-48). Generally, optimal results for the freezing of hematopoietic stem and progenitor cells can be achieved with a cooling rate of about 1-2.degree. C. per minute with a final storage temperature of about -70.degree. to about -196.degree. C. with other blood cells generally within these same ranges.
The successful recovery of human bone marrow cells after long-term storage in liquid nitrogen has been described (1983, American Type Culture Collection, Quarterly Newsletter 3(4):1). In addition, stem cells in bone marrow were shown capable of withstanding cryopreservation and thawing (Fabian, I., et al., 1982, Exp. Hematol, 10(1):119-122).
The widely accepted industry standard cryoprotectant for use in the cryopreservation of most cells, including whole blood, umbilical cord blood, bone marrow, granulocytes, neutrophils, platelets and hematopoietic stem and progenitor cells, is dimethyl sulfoxide (DMSO). This can generally be attributed to the widespread experience and knowledge of DMSO-based cryopreservation solutions and a general perception that DMSO provides superior protection and maximal cell viability. While DMSO is generally effective for these purposes, it is also known to be physiologically pernicious, particularly at higher concentrations, and thereby tends to irritate both those involved in the cryopreservation process and the patient into whom the cryopreserved cells are introduced with resultant hypertension, vomiting and nausea. The pernicious nature of DMSO is also thought to result in a lower in vitro viability of the cryopreserved cells.
Accordingly, a substantial need exists for a simple, physiologically compatible, alternative cryopreserving solution capable of providing a comparable level of cell viability both in vivo and in vitro.